Effects of acute heat stress on haemato-biochemical parameters, oxidative resistance ability, and immune responses of hybrid yellow catfish (pelteobagrus fulvidraco × P. vachelli) juveniles

This study investigated the effect of heat stress on the physiological parameters, oxidation resistance ability and immune responses in juvenile hybrid yellow catfish. Heat stress group exposed to 35 °C and control to 28 °C. Blood and liver were sampled at different hours’ post-exposure. Results showed that red blood cell (RBC), white blood cell (WBC) counts, Hemoglobin (HGB) levels and hematocrit (HCT) values increased significantly (P < 0.05) post-exposure to heat stress. This indicates the increase of cell metabolism. Serum alanine aminotransferase (ALT) and aspartate transaminase (AST) activities, total cholesterol (TC), total protein (TP), triglyceride (TG) and glucose increased significantly (P < 0.05) indicating the need to cope with stress and cell damage. Liver TC, TG, COR hormone, C3 complement increased significantly from 24 to 96 h. Heat stress mostly affects the hepatic antioxidant and immune resistance functions, resulting in increments of cortisol levels, lysozyme, superoxide dismutase (SOD), and catalase (CAT) enzyme activities. The increase of Malondialdehyde (MDA), alkaline phosphatase (AKP) indicate stimulation of the immune responses to protect the liver cells from damage. The decrease in Liver TP indicated liver impairment. Decrease in Glycogen content from 6 to 96 h indicated mobilization of more metabolites to cope with increased energy demand. Interestingly, results showed that heat stress trigged costly responses in the experimental fish like accelerated metabolism and deplete energy reserves, which could indirectly affect ability of fish to set up efficient long term defense responses against stress. These results provide insight into prevention and management of stress in juvenile hybrid yellow catfish.


Introduction
Several environmental stress elements affect homeostasis and can affect biological function to some extent (Dagoudo et al. 2021, Gracey et al. 2004. One such environmental stressors is water temperature, which has profound effects on physical and chemical processes within biological systems (Scott and Johnston 2012). These changing environmental factors lead to effects in the fish health by stress. Stress of the fish is nonspecific response and the fish need any demand made upon it. Stress in fish may be induced by various abiotic environmental factors such as changes in water temperature, pH, oxygen concentration and water pollutants including pesticides, insecticides (Dagoudo et al. 2021, Kandeepan 2014. Changes in water temperature affect many properties and functions of biomolecules and structural components of Missinhoun Dagoudo mdagoudo@gmail.com cells: including the assembly, folding, activity and stability of proteins structure and rigidity of lipids and fluidity and permeability of cell membranes (Pan et al. 2008). Fish that may not avoid these temperature fluctuations therefore are likely to be exposed to stressful conditions. Any change in culture water temperature may influence the survival, physiological functions and immune defenses of fish, such as Cyprinus carpio (Li et al. 2019), Oncorhynchus mykiss (Bowden 2008), Oryzias latipes (Lee et al. 2014). Appropriate temperatures promote growth and development of fish, while temperatures that exceed particular limits have negative effects (Rahman et al. 2019). Sudden changes in temperature can weaken the immune system and enhance susceptibility to pathogens; in extreme cases, high temperature may lead to shock and sudden death (Chen et al. 2002). Stress response involves many physiological changes with alteration in immune mechanisms and blood composition. Furthermore, Stress induces fluctuations in blood cell numbers and activities. An increase in red blood cell count and volume, and hemoglobin level usually has been reported in fish subjected to stress and decrease in white blood cell count, especially of lymphocytes usually occurs in fish subjected to stress (Kandeepan 2014).
On the other hand, these hematological parameters of fish can be used as indicators of physiological conditions and monitoring diseases and the stress caused by handling (Guillen et al. 2019). The study of blood indices in fishes has been extensively used for discovery on physio-pathological alterations in diverse conditions of stress. The stress reactions vary between the species, stage of development, also among individuals within the same species. Enzymes such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the ubiquitous aminotransferase in the mitochondria, can be released to the plasma following the tissue damage and organ dysfunction due to stress. Increasing findings indicate that environmental stressors can lead to an increase of the plasma ALT and AST activities in the fish, implying that both ALT and AST can serve as an indispensable indicator capable of monitoring changes in fish physiological parameters following the exposure to environmental stressors (Cheng et al. 2017). Studies of the immune response in yellow catfish have shown that high temperatures lead to stress, outbreaks of bacterial infection, and high mortality in summer (Liu et al. 2016).
Previous research studies have also evidently demonstrated that the ambient thermal stress can exert severe effects on fish, including malfunctioning of cell metabolism (Lannig et al. 2006), weakening of immune system and consequently a sharp decrease of disease resistances (Xu & Zhao 2013). Similarly, thermal stress can change the levels of various substances such as total protein (TP) and serum glucose (GLU) (Liu et al. 2016), decrease metabolic activities (Lu et al. 2016) as well as weaken non-specific immune defense system in fish (Qiang et al. 2013). Many blood biochemical indices change in fish for the reaction to the stress. For example, grass carp (Ctenopharyngodon idellus) exhibit significant declines in serum superoxide dismutase (SOD) and acute increases in serum glucose when subjected to high temperature stress (Cui et al. 2014). It was studied the effects of high temperature on different tissues of rainbow trout (Oncorhynchus mykiss) and reported that serum SOD and malondialdehyde (MDA) increase significantly under heat stress (Wang et al. 2016).
Yellow catfish (Pelteobagrus fulvidraco) is one of the most significant freshwater aquaculture species in China. It is one of the native freshwater species that has a strong market demand in southern and eastern China and is commonly called yellow bonefish by farmers (Dong et al. 2011). Production of yellow catfish has expanded because of widespread availability of fry, innovative feed technology, the species' tolerance for long distance transportation, and its high market value (Tang et al. 2012). Because of its tender flesh, few intermuscular spines, and delicious flavor, yellow catfish is quite popular with consumers in China (Liu et al. 2013).

Experimental animals and source
Juvenile hybrid Yellow catfish (Pelteobagrus fulvidraco♀ xPelteobagrus vachellii♂) were obtained from Yixing fish farm of Freshwater Fisheries Research Center (FFRC). The study was conducted at Freshwater Fisheries Research Center of Chinese Academy of Fishery Sciences, Wuxi, Jiangsu. Fish was fed on the commercial fish diet twice a day (08:30 Am and 04:30 Pm) 6 weeks at a rate of 5% wet body weight up to apparent satiation before the experimental treatments in a two-sided recirculating aquaculture system (RAS). During the experiment period, the water quality was maintained at normal pH 6.8-7.6, normal DO: 6.5-7.0 mg/L, NH 3 < 0.05 mg/L, H 2 S < 0.01 mg/L, with natural photoperiod, continuous aeration.

Experimental design
The experiment was carried out in six (6) plastic tanks (450 L) of the Recirculating Aquaculture System (RAS), in the green house of FFRC, south campus. Each tank contained 30 healthy, uniform sized (44.95 ± 0.04 g), randomly picked juvenile hybrid yellow catfish. Prior to the experiment, the juvenile fish was acclimatized to the experimental conditions and facilities with the commercial diet (38% crude protein, 5% crude lipid) in the plastic tanks for 14 days. After acclimatization, the fish was divided into two groups; the control group (CG) and Heat stress group (HG). The water temperature for the heat stress group was raised gradually up to 35 0 C using artificial temperature incubators at a constant rate (4 0 C /h) while that of the Control group was kept at 28 °C. Samples were chosen based on the guide for the care and use of laboratory animals in China. Sampling was conducted after; 0, 6, 12, 48 and 96 h. Three (3) fish were sampled for data collection per tank. Sampled fish were anesthetized in 0.05% tricaine methane sulfonate Sigma Diagnostics INS. and St. Louis,MO). The blood samples were drawn from the fish using sterilized syringes and used to measure the change of total hematological, serum biochemical parameters. In addition, the isolated liver samples were preserved at − 80 °C until use. During the heat stress experiment, there was no feeding at all and minimal human interference to prevent additional stress to the fish. Each treatment was tested in triplicate.

Determination of the hematological parameters
Blood samples were collected from the caudal vein of each of the sampled fish, using sterile disposable plastic syringes. Heparine sodium (1%) was used as an anticoagulant. The collected blood samples were immediately subjected to hematological analysis because long-term storage can modify the results of the analyses, probably due to storagerelated degenerative changes that may occur (Cheng et al. 2018). A Mindray-BC-5300 Auto Hematology Analyzer (Shenzhen, China) was used to measure Red blood cells counts (RBC), White blood cell counts (WBC), Hemoglobin concentration (Hb) and Hematocrit (Hct).

Measurement and determination of the serum biochemical parameters
The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), glucose (GLU), glycogen, cortisol, triglycerides (TG), Total Cholesterol (TC) and total protein (TP) was measured in the automatic biochemical analyzer (Mindray BS-400 bio-chemistry analyzer) using kits assay specially designed for fish detection. Glycogen content was estimated with anthrone method.

Measurement of the antioxidant enzymes and immune resistance in the liver of Yellow Catfish
The sampled fish was then dissected. The fish abdominal cavity was cut open instantly after blood sample. A piece of liver was stored at -40°C for routine analysis (Cheng et al. 2018). Liver samples were homogenized in an ice bath and diluted with ice-cold phosphate buffer solution (PBS) (1:10 dilution) with a liver extract and homogenate medium (pH 7.4) ratio of 1:9 (W: V), and then centrifuged for 20 min (10,000 r/min, 4°C) (Cheng et al. 2018). The supernatant was removed for analysis. The superoxide dismutase (SOD) activity, catalase (CAT) activity, and malondialdehyde (MDA) content, was measured by kits using the xanthine oxidase method, the colorimetric method and the thiobarbituric acid calorimetry respectively. Lysozyme (LZM), Complement (C3), were measured using enzymelinked immunosorbent assay (ELISA) test kits (Nanjing Jiancheng Biotechnology Co., Ltd, China). Alkaline phosphatase (AKP) were measures with the supernatant, the kits were purchased from Nanjing Jiancheng Biotechnology Research Institute (Nanjing, China).

Statistical analyses
The results were statistically analyzed using SPSS 20.0 for Windows by one-way ANOVA followed by Duncan's multiple range tests to examine the effect of high temperature stress at each water temperature. Data was presented as means plus or minus the standard error. P < 0.05 was fixed as the significance minimum level.

Effect of heat stress on the hematological parameters
The counts of RBC and WBC ( Fig. 1a & b) increased uniformly after 12 h in the heat stress was a significantly higher than control group at 24 h, 48 and 96 h (P < 0.05). The levels of hemoglobin (HGB) and hematocrit HCT ( Fig. 1c & d) increased significantly after 12 h for HGB and 6 h for HCT in the heat stress group, and then decreased slightly at 96 h. There was a significant difference in the concentrations of hemoglobin and hematocrit HCT between the control group and the heat stress group after 12 h, 24 h, 48 and 96 h (P < 0.05). There was no significant change in the hematological parameters of the control group during the experiment.

Effect of heat stress on serum biochemical parameters
The concentration of ALT increased sharply after 6 h attaining the highest significant amount in the heat stress group. Then it decreased uniformly after 12 h and remained fairly constant up to 96 h. ALT levels were significantly higher (P < 0.05) in the heat stress group than the control of TG was significantly (p < 0.05) higher in the heat tress group than in the control group after 6 h, 12 h, 24 h, 48 and 96 h. There was no observable change in the control group during the experiment.

Effect of heat stress on liver biochemical parameters
In the heat group, the concentration of Total protein (TP) (Fig. 3a) decreased rapidly after 6 h then remained significantly (P < 0.05) lower than that of the control group at 6 h, 12 h, 24 h, 48 and 96 h. The levels of total cholesterol (TC) (Fig. 3b) increased significantly (P < 0.05) and were higher than control group at 12 h, 24 h, 48 and 96 h. The concentration of TG ( Fig. 4c) increased significantly after 12 h then decreased after 24 h. The concentrations of TG were significantly higher after 12 h, 24 h, 48 h, and 96 h group at 6 h, 12 h, 24 h, 48 and 96 h (Fig. 2a). The concentration of AST ( Fig. 2b) in the heat stress group also increased sharply after 6 h and remained significantly (P < 0.05) higher than the control group at 6 h, 12, 24 h, 48 and 96 h. The concentration of serum TC and TP ( Fig. 2c  & d) in the heat stress group increased gently from 6 to 48 h, then reduced slightly after 96 h and was significantly (P < 0.05) higher than the control group at 6 h, 12 h, 24 h, 48 and 96 h. The concentration of serum glucose (GLU) (Fig. 2e) in the heat stress group increased sharply after 6 h, then decreased sharply up to 24 h, then remained fairly constant at 48 to 96 h and was significantly higher than control group (P < 0.05) at 6 h, 12, 24, 48 and 96 h. The levels of Total Triglycerides (TG) (Fig. 2f) increased sharply after 6 h, attaining maximum after 12 h, then decreased slightly from 24 to 48 h and slightly increased after 96 h. The level as mean ± SEM. Significant differences (P < 0.05) between heat and control groups at same sampling time are marked by asterisks (*)

Effect of heat stress on liver anti-oxidant enzymes and immune resistance
The level of MDA, Superoxide dismutase (SOD) and Catalase (CAT) enzyme activity (Fig. 4a, b & c) increased sharply after 6 h, then fairly increased further up to 48 h and finally reduced slightly after 96 h in the heat stressed group. MDA levels reached maximum after 48 h were significantly higher (P < 0.05) in the heat stress group from 6 h up to 96 h than the control group that showed no observable changes. There was a sharp increase in the lysozome activity (LZM) (Fig. 4d) after 6 h, followed by slight increase up 48 h and a slight decrease at 96 h, in the heat stress group. There was no change in the lysozome activity in the control group. The lysozome activity was significantly higher (P < 0.05) in the (P < 0.05). The concentration of cortisol hormone increased sharply after 6 h, (Fig. 3d) then uniformly up to 96 h and was significantly higher than control group (P < 0.05) at 12 h, 24, 48 and 96 h during the experiment. The C3 complement (Fig. 3e) increased in the heat stress group and was significantly higher (P < 0.05) than the control group from 6 h up to 96 h. The glycogen content (Fig. 3f) reduced significantly after 6 h, 12 h, 24 h, 48 and 96 h in the heat stress group. But there was no observable change in the control group. In this study, there was a significant increase in counts of RBC and WBC, concentration of hemoglobin (HGB) and the percentage of hematocrit (HCT) in the heat stress group. The concentration of RBC and HGB increased because the high water temperature increased the body metabolic rate in the experimental fish. Therefore, the fish responded by producing more red blood cells and hence hemoglobin so as to transport more oxygen and meet the increased demand for oxygen in the fish body to supply sufficient oxygen for the increased metabolic rate (Dagoudo et al. 2021). Also as the temperature increases, oxygen absorption by RBC decreases. Thus, the body compensates this reduction by increasing the amount of red blood cells in circulation. This was also reported by De et al. (2019) on the study on effects heat stress group than the control group. The concentration of Alkaline phosphatase (AKP) in the heat stress group rapidly increased after 6 h ( Fig. 4e) and then continued uniformly up to 48 h and finally there was a slight decrease after 96 h. The concentration of AKP was significantly higher (P < 0.05) in the heat stress group from 6 h up to 96 h.

Effect of heat stress on the hematological parameters
The study of hematological characteristics in cultured fish species is an important tool in the development of aquaculture system (Mauri et al. 2011). It is necessary to  (e) and Glycogen content (f) on liver of juvenile Yellow catfish Pelteobagrus fulvidraco × Pelteobagrus Vachelli. Note: Data are mean values of nine replicates expressed as mean ± SEM. Significant differences (P < 0.05) between Heat and control groups at same sampling time are marked by asterisks (*) the immune system, and changes in blood parameters can be used to assess the physiological health of fish (Li et al. 2010). WBCs are another important component of blood is which are involved in cellular immunity.
In the current study, increases in the numbers of WBCs showed that heat stress affected the immune function in the juvenile Yellow catfish. The increase in numbers of WBCs within this study hint at possible stimulation of the nonspecific immune system of the experimental fish. Similar results have been reported by Huang et al. (2018) who found a decrease in non-specific immunity after heat stress in Prenant's Schizothoracin (Schizothorax prenati). Therefore, the significant increase of WBC in the fish under heat stress (35 0 C) may be due to cope up the stress.
In this study the concentration of hematocrit (HCT) increased due to increase in the number of erythrocytes of water temperature and diet on blood parameters and stress levels in juvenile hybrid grouper. Hemoglobin (HGB) is the main oxygen-carrying protein in RBC, and affects the transport capacity of blood oxygen and the numbers and function of RBCs (Bao et al. 2018). Yellow catfish is a freshwater ectothermic fish and its metabolism is highly temperature-dependent (Avvakumov al. 2010). Furthermore, this study showed that the observed increases in RBCs are combined with increased levels of HGB to satisfy a higher oxygen demand needed for higher metabolic requirements under heat stress (Dagoudo et al. 2021). It also was noted that the RBC counts and HGB levels were decreased after 96 h of heat stress, probably because the fish anti-stress mechanism gradually weakened with time hence the slight decrease at 96 h. Bao et al. (2018), demonstrated similar changes in the GIFT tilapia. Blood is an important part of Note: Data are mean values of nine replicates expressed as mean ± SEM. Significant differences (P < 0.05) between Heat and control groups at same sampling time are marked by asterisks (*) very important transaminases that are transformed between catalytic amino acid and ketonic acid. In our study, when the activity of ALT and AST both increase under heat stress for 6 to 96 h, the damage by high temperature to the fish's liver cells may have caused the emission of ALT and AST, which is similar to the previous studies on catfish (Horabagrus brachysoma) and rainbow trout (Oncorhynchus mykiss). Therefore, the activities of serum AST and ALT can be used to monitor the health status of fish (Javed and Usmani 2019).
Serum TC is the sum of all cholesterol in the blood in the form of different lipoproteins, and the liver is the main organ for synthesis and storage of cholesterol (Komprda et al. 2014). In this current study, there was a significant increase in serum TC levels under heat stress in the juvenile yellow catfish. The changes in serum TC show that high temperature stress triggered some liver damage.
Similarly, in this study, serum TP increased significantly from 6 to 48 h in the heat stress group. Serum TP maintains the balance of plasma colloid osmotic pressure and pH, and functions in transportation, coagulation, immunity, and energy supply. Thus, increases in serum TP are due to decreased water content in serum (Li et al. 2013), which reflects the reduction in water content of blood and hence the concentration of blood during stress. This result also confirms the increase in blood density reflected in the HCT changes. Serum total protein levels can be used as a diagnostic tool and a valuable test for evaluating the general physiological state in fish.
Glucose is a significant nutrient in serum, which is controlled by the external and internal environment. In this, study, the rapid increase in the concentration serum glucose at 6 h in the heat stress group could have been due to mobilization of glucose from muscle and liver sources. When fish are exposed to heat stress, muscle and hepatic sources of glucose are rapidly mobilized which results in the sharp increase in serum glucose to supply the required energy in counteracting stress. It was reported that serum glucose levels in silver catfish (Rhamdia quelen) and Coilia ectenes Jordan (Coilia nasus) increase at high water temperature (Qiang et al. 2016). Stressful stimuli stimulate rapid secretion of both glucocorticoids and catecholamine from the adrenal tissue offish both hormones produce a rapid hyperglycemia. Frequent occurrence of accelerated metabolism during exposure to environmental stressors has been demonstrated in several studies about hyperglycemia during stress, because plasma glucose levels are positively correlated with metabolic rate.
In this current study, the level of serum TG increased significantly with increasing time duration (P < 0.05) in the heat stress group. The concentrations of TG increased with probably because the high temperatures accelerated metabolic rate of the fish thus causing a rise in the demand (Red blood cells) and level of hemoglobin or a decrease in plasma volume. The increased HCT enhances the function of RBCs within the normal range of viscosity, and the greater number of RBCs and higher HGB concentration promote the transport of oxygen (Bao et al. 2018). This explains why the changes in HCT in the heat stress group showed the same trend as the changes in concentration of Red Blood Cells (RBC) and hemoglobin. In other comparative studies, the seawater flathead grey mullet (Mugil cephalus) and the freshwater goldfish (C. auratus) presented significant hematological variations. Higher values of RBC and HCT, were reported in the grey mullet in respect to goldfish (Parrino et al. 2018).

Effect of heat stress on serum biochemical parameters
Serum biochemical parameters, such as serum glucose concentration, ALT, AST activity and TP & TC levels, are related to temperature stress in fish (Liu et al. 2016). Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) in the mitochondria can be released to the plasma following the tissue damage and organ dysfunction. Several studies have shown that changes in the activities of ALT and AST in serum usually directly indicate cell damage in specific organs, because the two aminotransferases are present in mitochondria and can be released into the serum as a result of tissue damage and organ malfunction (Liu et al. 2016).
The increased activities of ALT and AST observed under heat stress group treatment in this study indicate that the enhanced ALT and AST activities could be due high water temperature stress. Several findings have showed that environmental stressors can lead to an increase of the plasma ALT and AST activities in the fish, implying that both ALT and AST can serve as an indispensable indicator capable of monitoring the changed fish physiology following the exposure to environmental stressors such as high water temperature (Li et al. 2019). In this study, ALT and AST levels significantly increased in the heat stress group from 6 h up to 96 h. Thus, the increased levels of AST and ALT activities in blood plasma at higher temperature indicated that organ dysfunction occurred in the experimental fish under heat stress. Similarly, previous studies indicate that a sharp increase of ALT and AST is observed in bream after thermal stress .
ALT is an important liver enzyme that reflects liver damage (Senior 2012). In this study, ALT activity increased significantly from 6 to 96 h. When liver and myocardial cells are impaired or when their permeability rises, AST and ALT are released into the blood, leading to the increase of blood transaminase activity. The ALT and AST are both the animals' nutritional status and indirectly reflects their nonspecific immune status (Huang et al. 2018).
This study also showed that heat stress induced a significant increase in the liver TG after 24, 48, and 96 h, which was probably due to the mobilization of lipid reserves to cope with an increased energy demand in fish (Stress et al. 2005). Similar results were demonstrated on the effect of thermal stress on the biochemical parameters in Pufferfish (Takifugu obscurus) (Cheng et al. 2018).
One of the first responses to environmental stressors, such as high water temperature, is the release of stress hormones like Adrenaline, Noradrenaline and cortisol. The release of these hormones especially Cortisol, triggers a range of biochemical changes in the fish body known collectively as secondary stress responses. Their metabolic effects may include hyperglycemia and depletion of glycogen tissue reserves. The catabolic effects catecholamines and corticosteroids on the energy reserves stored in the body tissues may result in reduced growth in stressed fish. The changes seen in the muscle and liver tissue agree well with the general picture of secondary responses, whereas the catecholamine are thought to cause the initial elevation in plasma glucose levels by mobilizing the glycogen reserves (glycogenolysis) corticosteroids may contribute to the maintenance of hyperglycemia via the stimulation of gluconeogenesis. The primary stress reaction in fish are characterized by raised levels of stress hormones, cortisol and catecholamine in the blood (Dagoudo et al. 2021). Cortisol is the most commonly used indicator of stress in fish (Saravanan et al. 2011). In the present study, the levels of cortisol hormone increased and rose higher in the heat stress group than the control group. The releases of these catecholamines and cortisol trigger a broad collection of biochemical changes known collectively as secondary stress responses. The metabolic effects may include hyperglycemia and depletion of glycogen tissue reserves. The catabolic effects catecholamine and corticosteroids on the energy reserves stored in the body tissues may result in reduced growth in stressed fish. Catecholamines deplete glycogen reserves in fish liver and muscle (Kandeepan 2014).
To meet the increased energy request of stressed animals, glycogen, due its accessibility for energy production, is speedily catabolized leading to the huge losses of this energy reserves. In this study, it was noted that the exposure of fish to heat stress resulted in a decrease in glycogen levels in the liver. Reduction in glycogen content of liver observed in the present study supports this view. Catecholamines deplete glycogen reserves in fish. Therefore, the observed glycogenolysis in the liver in this study after exposure to stress could possibly have been caused by a stress induced increase in circulating catecholamine. The depletion in glycogen stores should be Stress induced breakdown of for more energy of the juvenile yellow catfish. Therefore, the observed significant increase in the serum TG, was probably due to the mobilization of lipid reserves to cope with an increased energy demand in the juvenile yellow catfish under heat stress (Stress et al. 2005). Acute and chronic stress is typically associated with increased metabolic rate. The slight decrease in TG levels at 12 h was probably to inhibition of lipid metabolism by high temperature. Caijuan (Li et al. 2019), similarly demonstrated that the concentration of TG decreased with increasing temperatures, suggesting that high temperatures may inhibit the lipid metabolism of pikeperch. It was noticed that the concentration of serum glucose, TP, ALT activity, and TC slightly decreased at the end of the experiment. This could be that the fish could no longer adapt their physiology in response to high temperature stress by the end of the experiment.

Effect of heat stress on the liver biochemical parameters
In vertebrates, stress negatively affects body homeostasis and triggers a series of physiological and immune responses, with liver playing a key vital role. The Liver responds with changed metabolism, enabling the stressed animal to cope with the stress situation, which involves carbohydrate and lipid mobilization. The liver of fish is a sensitive indicator of metabolic adaptation to varying environmental influences such as high water temperature.
The total protein is a measure of the total amount of two classes of proteins found in the fluid portion of blood, albumin and globulin. Albumin protein which accounts for half of the total protein found in blood plasma helps to prevent fluid from leaking out of blood vessels. It also regulates the osmotic pressure in the plasma to prevent water from leaking out of the blood vessels. While globulins are an important part of the immune system. Total Proteins are essential for overall health of fish. During stress the immune activity decreases in the fish body.
In this study, a significant decrease in the levels of Liver TP was observed in the Heat stress group after 12 h up to 96 h during the experiment. The significant decrease (P < 0.05) in liver protein level might be due to impaired protein synthesis caused by liver disorder during heat stress. The TP concentrations decreased with time at high temperature (35 0 C). The decline in the concentrations of TP could have been caused by changes in the structure of the fish liver, thereby reducing aminotransferase activity, decreasing deamination capacity, and impairing the control of fluid balance (Coz-Rakovac et al. 2005). Total serum protein is also used as an indicator of liver impairment (Firat and Kargin 2010). The TP content of fish reflects this multiplicity in complement proteins helps to expand their innate immune recognition capacity and response. Understanding the functions of complement in fish and the roles the individual proteins, including the various isoforms, play in host defense, is important not only for understanding the evolution of this system but also for the development of new strategies in fish health management (Holland and Lambris 2002).

Effect of heat stress on liver anti-oxidant enzymes and immune resistance
Superoxide dismutase (SOD) is considered to play a key role in the first step of the enzymatic anti-oxidative defense system (Dagoudo et al. 2021). SOD activity helps the organism to control the intracellular steady status level of superoxide (O 2 -) which is a relatively strong oxidizing agent. In the present study, there was significant increase in SOD activity in the liver of the juvenile catfish subjected to heat stress under high temperature of 35 0 C. This demonstrates that SOD provides effective protection to cells against heat shock in yellow catfish. Lushchak and Bagnyukova (2006) demonstrated that SOD activity significantly increased in the brain, liver and kidney after 12 h exposure to high temperature (35 0 C) in goldfish (Carassius auratus). Parihar et al. (1996) found that SOD activity in the gills of the freshwater catfish (Heteropneustes fossilis) increased significantly at 32 0 and 37 0 C after 1-4 h compared with the control (25 0 C). It can be inferred from these results that fish can protect their cells from ROS damage by increasing SOD activity during heat stress (Wang et al. 2016).
Catalase (CAT) is one of the most efficient antioxidant enzymes found in cells. It catalyzes the reaction by which hydrogen peroxide is decomposed to water and oxygen. Hydrogen peroxide is a hypothetically dangerous species because of its ability to easily cross biological membranes and high stability. CAT has the ability to eliminate hydrogen peroxide (H 2 O 2 ), which helps to counteract the influence of oxidative stress (Duan et al. 2015). In this present study, there was a significant increase in the CAT activity in the heat stress group. The significant increase in the activity of CAT in liver could have been for protection against lipid hydro peroxides and H 2 O 2 in the fish liver under heat stress. The increased rates of mitochondrial respiration caused by stress could have enhanced the formation of ROS, and hence increased the initiation of SOD and CAT at translational and transcriptional levels against oxidative damage Madeira et al. 2013) also demonstrated that Catalase activity significantly increased in L. ramada, subjected under thermal stress. The antioxidant enzymes are the first intracellular defense against oxidative stress and they carbohydrate pool, supplies the growing energy requirement to meet the stress condition in general accompanied by an increase in glucose content as observed in this study (Kandeepan 2014). The depletion in glycogen supplies should be supplemented by an increase in glucose content as perceived in this study. The abrupt decline in liver glycogen in the heat stress group resulted in the hyperglycemia of the blood. This consistent decrease in glycogen reserves suggests that glycogenesis was impaired. Murugaian (2008) also observed that the glycogen content in the liver and muscle decreased with increasing temperatures and with more exposure periods of thermal stress. Because of the stress, the fish makes suitable adjustments for which the stored energy is utilized. This may be the reason for the decreased amount of glycogen content (Wu et al. 2015). The glycogen content was observed in the decreasing order with time under heat stress. Because of the stress, the fish makes suitable adjustments for which the stored energy is utilized. This may be the reason for the decreased amount of glycogen content.
Complement system (C3) is an important component of the immune defense of fish. It plays an important role in the immune defense against the inflammation and the bacterial invasion. C3 is the key component of both classical and lectin pathways responsible for various immune effector functions (Holland and Lambris 2002). Previous studies show that the C3 mRNA expression level can increase significantly in response to environmental stressors (Qi et al. 2011). In addition, the complement activity can be considered as an essential indicator of immune-competence in fish under the exposure to stressors. In this study the levels of C3 increased gradually, suggesting that the complement system was activated by thermal stress. Complement, an important component of the innate immune system, is comprised of about 35 individual proteins. Activation of complement results in the generation of activated protein fragments that play a role in microbial killing, phagocytosis, inflammatory reactions, immune complex clearance, and antibody production (Qi et al. 2011). Fish seem to possess activation pathways like those in mammals, and the fish supplement proteins identified thus far show various homologies to their mammalian counterparts. Because information about supplement proteins, complement receptors and regulatory proteins in fish is far from complete, it is uncertain whether all the supplement functions that have been known in mammals also occur in fish. However, it has been clearly demonstrated that fish complement can lyse foreign cells and opsonize foreign organisms for destruction by phagocytes. There are also signs that supplement fragments participate in inflammatory responses. Fish possess several isoforms of numerous complement proteins, such as C3 and factor B. It has been assumed that the function of parameters like; counts of RBC and WBC, concentration of hemoglobin (HGB) and the percentage of hematocrit (HCT) in the heat stress group. There were also changes in blood and live biochemical indices like increased enzyme activity (ALT and AST), TP, TC, TG, glucose and glycogen. The initial rapid rise in hematological parameters RBC, HGB and HCT) indicated increased cell metabolism and while the final decline indicated cell damage. Changes in WBC, TG, TC, AKP, C3 complement and antioxidants indicated that heat stress affected the immunity system. In addition, the results showed that high temperature trigged costly stress responses in the experimental fish like accelerated metabolism and deplete energy reserves, which could indirectly affect ability of fish to set up efficient long term defense responses against stress. The results of this study provide insight into prevention and management of stress in juvenile hybrid Yellow Catfish.

Supplementary Information
The online version contains supplementary material available at https://doi.org/10.1007/s11259-022-10062-1. Data Availability and material statement The data for this work will be made available when needed and on request. regulate redox-dependent signaling, which is indispensable for innate immunity (Dixon et al. 2012).

Conflict of interest
Malondialdehyde (MDA) is a cytotoxic end-product of lipid peroxidation, so the MDA content indicates the extent of oxidative damage (Mancino et al. 2011). In this study a significant increment in the MDA concentrations was observed at 6, 12, 24 and 48 h after high temperature stress showing that heat stress caused oxidative stress, which probably led to injury of liver cells. These results further suggest that heat stress worsens oxidative stress by generating ROS. This occurrence could be due to the capacity of high temperature to denature and damage antioxidant enzymes (Abele and Puntarulo 2004), reduce antioxidant defenses and disturb physiological homeostasis (Stress et al. 2005), leading to lipid peroxidation injury in the liver of the juvenile yellow cat fish (Parihar et al. 1996).
Lysozyme enzyme is a critical innate immunity component in maintaining immune defense system to prevent bacterial infection by destroying the peptidoglycan layer of predominant gram-positive bacteria and some gram-negative bacteria. Lysozyme activity is stimulated to improve the immune defense by inducing alterations in immune-regulatory functions when pathogenic bacteria and various stress-induced substances attack the fish (Kong et al. 2012). In this present study, the lysosome activity increased after increased significantly after 6 h up to 96 h in the heat stress group. Ndong et al. (2007), demonstrated that lysozyme activity increased significantly when Tilapia fish (Oreochromis mossambicus) transferred to 31 and 35 °C for over 48-96 h. Also Guardiola et al. (2015) reported a substantial increase in the lysozyme activity of gilthead seabream, (Sparus aurata) exposed to arsenic, cadmium, and mercury.
Alkaline phosphatase (AKP) is a vital enzyme which performs a significant role in phosphorus metabolism in the fish organs and tissues. In this study, the activities of AKP significantly increased in the heat stress group, indicating that the increased level of AKP might result from the disturbances in both physiological and functional mechanisms under heat stress. Thus, taken together, heat stress can exhibit an adverse effect on the innate immunity by inducing oxidative damage to macromolecules and disrupt the physiology by regulating the activities of metabolic and immune-related enzymes (Cheng et al. 2017).

Conclusion
The Results from this study showed that high temperature stress caused changes in the physiological parameters and induced immune responses of juvenile hybrid Yellow catfish. There was a significant increase in hematological of gilthead seabream (Sparus aurata). Fish Shellfish Immunol 45 (1) (3)